stomatal anatomy microscopy imaging

1. Prepare

1.1 Find a mold folder and take out one working page.Need to determine if there is two finish working color markers. If only one, need to finish it priority.

1.2 Open the data sheet and find the number of the page.

For North quantity test: https://docs.google.com/spreadsheets/d/166n-3RT2ai1lQwvxABFdsAe6WKWF4I5sx1N6Zqs4soU/edit?usp=drive_link

For South quantity test: https://docs.google.com/spreadsheets/d/1xihtO0SSQLMLCAdFZAFwq3XT1wGSoP9B/edit?usp=drive_link&ouid=102207485550371806190&rtpof=true&sd=true

For north garden: https://docs.google.com/spreadsheets/d/1xihtO0SSQLMLCAdFZAFwq3XT1wGSoP9B/edit?usp=sharing&ouid=102207485550371806190&rtpof=true&sd=true

For south garden: https://docs.google.com/spreadsheets/d/1l0IMaZir0SRKR14aO3ckYuN5QF2ONtCX/edit?usp=drive_link&ouid=102207485550371806190&rtpof=true&sd=true

1.3 Take an unlabeled grid sheet and copy the sample IDs and any notes (most sheets will not have notes) from the labeled grid sheet to the unlabeled grid sheet.

1.4 Put the tray on the table, the label should be on the upper left corner.

1.5 Move the samples from the labeled grid sheet to the preparation area in the metal pallet in the same order. Only move one sample at each time, and put them facing up.

1.6 Check that the sample is clean and clean with paper towels if needed. If needed, can use ethanol to clean the surface, but need to use the tissue to dry it before the next step.

1.7 Add the number ID on the data sheet * YYYY_region_row_col_surface (if doing a single image per mold) * YYYY_region_row_col_surface_rep (if doing a multiple images per mold) * Use _, no whitespace * Always use lowercase * col – always use three digits

Examples:

• 2023_north_101_001_upper
• 2023_north_101_001_upper_1

2.Nail Polish

2.1 Open a bottle of nail polish and make sure the brush is clean. Make sure close it if don’t use it.

2.2 Apply an even coat of nail polish to each mold and put them back to the working place (full cove the mold, and as thin as possible).

Close the nail polish cover when finish used.

2.3 Wait for drying, about 10 minutes.

2.4 Apply the dried nail polish with transparent tape and remove it from the mold (The tape could be a little longer than tape. Fold the bottom end, so it will be easy to remove it later.)

2.5 Apply tape and nail polish to the slide, make it as flat as possible. Use a black marker to carefully record the sample number on the tape. Example:

• 001_001_up
• 001_001_low

3. Use a microscope to take stomatal images.

3.1. Computer and microscope

3.1.1 If the microscope has a cover, remove it carefully.

3.1.2 Open the light of the microscope (check the light cover is full open)

3.1.3 Move the side lever to the middle position

3.1.4 Open the console power.

3.1.5 Turn the objective lens to 10X.

3.1.6 The computer should be in sleep mode, If the screen is black, check if the monitor is on.

3.1.7 Open the NIS-Elements soft work on the computer.

3.2. Observation and Imaging

3.2.1 Place the prepared slide on the observation stage

3.2.2 Use the live Quality (The button on the right with a green arrow and camera). If this button not work, try live observation (The left one.)

3.2.3 Before the first time use. Make sure the objective lens magnification is match (Click once 10X) (If forget this, it may not affect the picture quality, but it will affect the background information.)

3.2.4 Open the AE control if not (Auto light control)

3.2.5 Adjust focus and position to find the right, clear image

• Easy to see the stomata
• The image should be bright, clear, and accurate focus
• Not a lot of bubbles or trichomes

3.2.6 Set resolution to \(1280 \times 960\) (check this when app close)

* Go to the Acquire/Camera Setting/Quality menu
    

3.3 Use capture button take photo

3.3.1 Use file label save image to the computer/desktop/Muir lab

3.3.2 The file format is .tif

3.3.3 Name the file

• YYYY_region_row_col_surface (if doing a single image per mold)
• YYYY_region_row_col_surface_rep (if doing a multiple images per mold)
• Use _, no whitespace
• Always use lowercase
• col – always use three digits

Examples:

• 2023_north_101_001_upper
• 2023_north_101_001_upper_1

4. Upload files and clean up

4.1 Upload the all images to the folder (Checking the photo size when upload it. 1280 should be 3.5-4MB)

For north quantity test: https://drive.google.com/drive/folders/1rGSGvlBm00Ye6gKyBNPd3TnKy1ifnGRN?usp=drive_link

For south quantity test: https://drive.google.com/drive/folders/1V32gC-TNtD1upgCEnvv6cIf-TG_ULzSN?usp=drive_link

For other data:

North 2023: https://drive.google.com/drive/folders/1fJWbtAW6wvPeD328JuI9pe7DQHdxnZ9G?usp=drive_link

South 2023: https://drive.google.com/drive/folders/187uVNJVR5PKVXMIEKz7vrJgWXw_dd2EQ?usp=drive_link

4.2 Report the image information on the data file, include the individuals number and, name abbreviation, and note if neeed.

Examples:

*2023_north_101_001_upper_1 | Frank | the image need to retake.

If take multiple photos for one sample. Just record every one by using different rep number.

4.3 Place the completed molds on the new gridded sheet (with IDs and notes), secure them with tape again, and put it back to the folder.

4.4 When finish one page, need to make the marker on the top. Use yellow highlighters for the front and red highlighters for the back side.

4.5 Remove the tape and clean the slide with ethanol for next time.

4.6 Used tape can be thrown directly into the trash can

4.7 When done for the day, put all slides back to the box

4.8 Close the computer console, turn off the microscope light, move the side lever to back, and put the cover back.

Precautions

If the microscope is not used for a long time, turn off the power

Don’t disturb other people’s work.

Some bad exmaple

1. This nail polish is too thin and unequal

2. The light cover is not full open.